DNA-based Tension Research Articles

Nuclease-Resistant L-DNA Tension Probes Enable Long-Term Force Mapping of Single Cells and Cell Consortia.

DNA-based tension probes with precisely programmableforce response provide important insights into cellularmechanosensing. However, their degradability in cell culture limitstheir use for long-term imaging, for instance, when cells migrate,divide, and differentiate. This is a critical limitation for providinginsights into mechanobiology for these longer-term processes. Here,we present DNA-based tension probes that are entirely designedbased on the stereoisomer of biological D-DNA, i.e., L-DNA. Wedemonstrate that L-DNA tension probes are essentially indestructibleby nucleases and provide days-long imaging without significant lossin image quality. We also show their superiority already for shortimaging times commonly used for classical D-DNA tension probes.We showcase the potential of these resilient probes to image minutemovements, and for generating long term force maps of single cellsand of collectively migrating cell populations.

Develop Tandem Tension Sensor to Gauge Integrin-Transmitted Molecular Forces.

DNA-based tension sensors have innovated the imaging and calibration of mechanosensitive receptor-transmitted molecular forces, such as integrin tensions. However, these sensors mainly serve as binary reporters, only indicating if molecular forces exceed one predefined threshold. Here, we have developed tandem tension sensor (TTS), which comprises two consecutive force-sensing units, each with unique force detection thresholds and distinct fluorescence spectra, thereby enabling the quantification of molecular forces with dual reference levels. With TTS, we revealed that vinculin is not required for transmitting integrin tensions at approximately 10 pN (piconewtons) but is essential for elevating integrin tensions beyond 20 pN in focal adhesions (FAs). Such high tensions have emerged during the early stage of FA formation. TTS also successfully detected changes in integrin tensions in response to disrupted actin formation, inhibited myosin activity, and tuned substrate elasticity. We also applied TTS to examine integrin tensions in platelets and revealed two force regimes, with integrin tensions surpassing 20 pN at cell central regions and 13-20 pN integrin tensions at the cell edge. Overall, TTS, especially the construct consisting of a hairpin DNA (13 pN opening force) and a shearing DNA (20 pN opening force), stands as a valuable tool for the quantification of receptor-transmitted molecular forces within living cells.

Using DNA-Based Tension Probes to Assess Receptor Forces Applied by Immune Cells

Using DNA-Based Tension Probes to Assess Receptor Forces Applied by Immune Cells

Using DNA-Based Tension Probes to Assess Receptor Forces Applied by Immune Cells

Using DNA-Based Tension Probes to Assess Receptor Forces Applied by Immune Cells

Molecular Force Imaging Reveals That Integrin-Dependent Mechanical Checkpoint Regulates Fcγ-Receptor-Mediated Phagocytosis in Macrophages.

Macrophages are a type of immune cell that helps eliminate pathogens and diseased cells. Recent research has shown that macrophages can sense mechanical cues from potential targets to perform effective phagocytosis, but the mechanisms behind it remain unclear. In this study, we used DNA-based tension probes to study the role of integrin-mediated forces in FcγR-mediated phagocytosis. The results showed that when the phagocytic receptor FcγR is activated, the force-bearing integrins create a "mechanical barrier" that physically excludes the phosphatase CD45 and facilitates phagocytosis. However, if the integrin-mediated forces are physically restricted at lower levels or if the macrophage is on a soft matrix, CD45 exclusion is significantly reduced. Moreover, CD47-SIRPα "don't eat me" signaling can reduce CD45 segregation by inhibiting the mechanical stability of the integrin barrier. These findings demonstrate how macrophages use molecular forces to identify physical properties and combine them with biochemical signals from phagocytic receptors to guide phagocytosis.

Exploring Integrin-Mediated Force Transmission during Confined Cell Migration by DNA-Based Tension Probes.

Mechanical forces have profound effects on the morphology and migration of cells in a two-dimensional environment. However, cells in vivo mostly migrate in three-dimensional space while physically constrained, and the mechanism by which cellular dynamic forces drive migration in this confined environment is unclear. Here, we present a method of fabricating microfluidic chips with integrated DNA-based tension probes to measure spatiotemporal variations in integrin-mediated force exerted during confined cell migration. Using this developed device, we measured the spatial locations, magnitudes, and temporal characteristics of integrin-ligand tension signals in motile cells in different microchannels and found that cells exerted less force and underwent increasingly transitory integrin-ligand interactions when migrating in confined spaces. This study demonstrates that the described method provides insights into understanding the migratory machinery of cells in geometrically confined environment that better mimics physiological conditions.

ST6Gal-I–mediated sialylation of the epidermal growth factor receptor modulates cell mechanics and enhances invasion

Heterogeneity within the glycocalyx influences cell adhesion mechanics and signaling. However, the role of specific glycosylation subtypes in influencing cell mechanics via alterations of receptor function remains unexplored. It has been shown that the addition of sialic acid to terminal glycans impacts growth, development, and cancer progression. In addition, the sialyltransferase ST6Gal-I promotes epidermal growth factor receptor (EGFR) activity, and we have shown EGFR is an ‘allosteric mechano-organizer’ of integrin tension. Here, we investigated the impact of ST6Gal-I on cell mechanics. Using DNA-based tension gauge tether probes of variable thresholds, we found that high ST6Gal-I activity promotes increased integrin forces and spreading in Cos-7 and OVCAR3, OVCAR5, and OV4 cancer cells. Further, employing inhibitors and function-blocking antibodies against β1, β3, and β5 integrins and ST6Gal-I targets EGFR, tumor necrosis factor receptor, and Fas cell surface death receptor, we validated that the observed phenotypes are EGFR-specific. We found that while tension, contractility, and adhesion are extracellular-signal-regulated kinase pathway-dependent, spreading, proliferation, and invasion are phosphoinositide 3-kinase-Akt serine/threonine kinase dependent. Using total internal reflection fluorescence microscopy and flow cytometry, we also show that high ST6Gal-I activity leads to sustained EGFR membrane retention, making it a key regulator of cell mechanics. Our findings suggest a novel sialylation-dependent mechanism orchestrating cellular mechanics and enhancing cell motility via EGFR signaling.

Single-Molecule Force Imaging Reveals That Podosome Formation Requires No Extracellular Integrin-Ligand Tensions or Interactions

Podosomes are integrin-mediated cell adhesion units involved in many cellular and physiological processes. Integrins likely transmit tensions critical for podosome functions, but such force remains poorly characterized. DNA-based tension sensors are powerful in visualizing integrin tensions but subject to degradation by podosomes which ubiquitously recruit DNase. Here, using a DNase-resistant tension sensor based on a DNA/PNA (peptide nucleic acid) duplex, we imaged podosomal integrin tensions (PIT) in the adhesion rings of podosomes on solid substrates with single molecular tension sensitivity. PIT was shown to be generated by both actomyosin contractility and actin polymerization in podosomes. Importantly, by monitoring PIT and podosome structure in parallel, we showed that extracellular integrin-ligand tensions, despite being critical for the formation of focal adhesions, are dispensable for podosome formation, as PIT reduction or elimination has an insignificant impact on structure formation and FAK (focal adhesion kinase) phosphorylation in podosomes. We further verified that even integrin-ligand interaction is dispensable for podosome formation, as macrophages form podosomes normally on passivated surfaces that block integrin-ligand interaction but support macrophage adhesion through electrostatic adsorption or Fc receptor-immunoglobin G interaction. In contrast, focal adhesions are unable to form on these passivated surfaces.

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay.

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

A reversible shearing DNA probe for visualizing mechanically strong receptors in living cells.

In the last decade, DNA-based tension sensors have made significant contributions to the study of the importance of mechanical forces in many biological systems. Albeit successful, one shortcoming of these techniques is their inability to reversibly measure receptor forces in a higher regime (that is, >20 pN), which limits our understanding of the molecular details of mechanochemical transduction in living cells. Here, we developed a reversible shearing DNA-based tension probe (RSDTP) for probing molecular piconewton-scale forces between 4 and 60 pN transmitted by cells. Using these probes, we can easily distinguish the differences in force-bearing integrins without perturbing adhesion biology and reveal that a strong force-bearing integrin cluster can serve as a 'mechanical pivot' to maintain focal adhesion architecture and facilitate its maturation. The benefits of the RSDTP include a high dynamic range, reversibility and single-molecule sensitivity, all of which will facilitate a better understanding of the molecular mechanisms of mechanobiology.

Super-resolved visualization of single DNA-based tension sensors in cell adhesion

Cell-extracellular matrix sensing plays a crucial role in cellular behavior and leads to the formation of a macromolecular protein complex called the focal adhesion. Despite their importance in cellular decision making, relatively little is known about cell-matrix interactions and the intracellular transduction of an initial ligand-receptor binding event on the single-molecule level. Here, we combine cRGD-ligand-decorated DNA tension sensors with DNA-PAINT super-resolution microscopy to study the mechanical engagement of single integrin receptors and the downstream influence on actin bundling. We uncover that integrin receptor clustering is governed by a non-random organization with complexes spaced at 20–30 nm distances. The DNA-based tension sensor and analysis framework provide powerful tools to study a multitude of receptor-ligand interactions where forces are involved in ligand-receptor binding.

Diffraction-Limited Mapping of Integrin Force Vector Fields with Valid-Mfm

Forces exerted through receptor proteins at the cell-surface interface regulate a wide variety of essential processes in mammalian cells, including stiffness sensing, cell migration, immune recognition, and coagulation. Molecular tension fluorescence microscopy (MTFM), has emerged as a powerful approach for measuring and mapping these force with diffraction-limited resolution. The most sensitive variant of MTFM relies on DNA-based tension probes, which consist of DNA hairpins flanked by fluorophore-quencher pairs that de-quench upon force-induced opening of the hairpin (DOI: 10.1038/ncomms6167).Unfortunately, MTFM does not inherently report on force orientation because signal is reliant on the orientation-independent distance between the probes’ two chromophores.

Recent Advances in Cell Adhesive Force Microscopy.

Cell adhesive force, exerting on the local matrix or neighboring cells, plays a critical role in regulating many cell functions and physiological processes. In the past four decades, significant efforts have been dedicated to cell adhesive force detection, visualization and quantification. A recent important methodological advancement in cell adhesive force visualization is to adopt force-to-fluorescence conversion instead of force-to-substrate strain conversion, thus greatly improving the sensitivity and resolution of force imaging. This review summarizes the recent development of force imaging techniques (collectively termed as cell adhesive force microscopy or CAFM here), with a particular focus on the improvement of CAFM’s spatial resolution and the biomaterial choices for constructing the tension sensors used in force visualization. This review also highlights the importance of DNA-based tension sensors in cell adhesive force imaging and the recent breakthrough in the development of super-resolution CAFM.

DNA Probes that Store Mechanical Information Reveal Transient Piconewton Forces Applied by T Cells

The advent of molecular tension probes for real-time mapping of piconewton forces in living systems has had a major impact on mechanobiology. For example, DNA-based tension probes have revealed roles for mechanics in platelet, B cell, T cell, and fibroblast function. Nonetheless, imaging short-lived forces transmitted by low-abundance receptors remains a challenge. This is a particular problem for mechanoimmunology where ligand-receptor bindings are short lived, and a few antigens are sufficient for cell triggering. Herein, we present a mechano-selection strategy that uses locking oligonucleotides to preferentially and irreversibly bind DNA probes that are mechanically strained over probes at rest. Thus, infrequent and short-lived mechanical events are tagged. This strategy allows for integration and storage of mechanical information into a map of molecular tension history. Upon addition of unlocking oligonucleotides that drive toehold-mediated strand displacement, the probes reset to the real-time state, thereby erasing stored mechanical information. As a proof of concept, we applied this strategy to study OT-1 T cells, revealing that the T cell receptor (TCR) mechanically samples antigens carrying single amino acid mutations. Such events are not detectable using conventional tension probes. Each mutant peptide ligand displayed a different level of mechanical sampling and spatial scanning by the TCR that strongly correlated with its functional potency. Finally, we show evidence that T cells transmit pN forces through the programmed cell death receptor-1 (PD1), a major target in cancer immunotherapy. We anticipate that mechanical information storage will be broadly useful in studying the mechanobiology of the immune system.

Peptide nucleic acid based tension sensor for cellular force imaging with strong DNase resistance

DNA is a versatile biomaterial with well-defined mechanical and biochemical properties. It has been broadly adopted to synthesize tension sensors that calibrate and visualize cellular forces at the cell-matrix interface. Here we showed that DNA-based tension sensors are vulnerable to deoxyribonucleases (DNases) which cells may express on cell membrane or secret to the culture environment. These DNases can damage the sensors, lower signal-to-noise ratio or even produce false signal in cellular force imaging. To address this issue, we tested peptide nucleic acid (PNA), chemically modified RNA and their hybrids with DNA as alternative biomaterials for constructing tension sensors. Four duplexes: double-stranded DNA (dsDNA), PNA/DNA, dsRNA (modified RNA) and PNA/RNA, were tested and evaluated in terms of DNase resistance, cellular force imaging ability and material robustness. The results showed that all PNA/DNA, dsRNA and PNA/RNA exhibited strong resistance to both soluble DNase I and membrane-bound DNase on cells. However, PNA/RNA-based tension sensor had low signal-to-noise ratio in cellular force imaging, and dsRNA-based tension sensor exhibited strong non-specific signal unrelated to cellular forces. Only PNA/DNA-based tension sensor reported cellular forces with highest signal-to-noise ratio and specificity. Collectively, we confirmed that PNA/DNA hybrid is an accessible material for the synthesis of DNase-resistant tension sensor that retains the force-reporting capability and remains stable in DNase-expressing cells. This new class of tension sensors will broaden the application of tension sensors in the study of cell mechanobiology.

DNA probes that store mechanical information reveal transient piconewton forces applied by T cells

The advent of molecular tension probes for real-time mapping of piconewton forces in living systems has had a major impact on mechanobiology. For example, DNA-based tension probes have revealed roles for mechanics in platelet, B cell, T cell, and fibroblast function. Nonetheless, imaging short-lived forces transmitted by low-abundance receptors remains a challenge. This is a particular problem for mechanoimmunology where ligand-receptor bindings are short lived, and a few antigens are sufficient for cell triggering. Herein, we present a mechanoselection strategy that uses locking oligonucleotides to preferentially and irreversibly bind DNA probes that are mechanically strained over probes at rest. Thus, infrequent and short-lived mechanical events are tagged. This strategy allows for integration and storage of mechanical information into a map of molecular tension history. Upon addition of unlocking oligonucleotides that drive toehold-mediated strand displacement, the probes reset to the real-time state, thereby erasing stored mechanical information. As a proof of concept, we applied this strategy to study OT-1 T cells, revealing that the T cell receptor (TCR) mechanically samples antigens carrying single amino acid mutations. Such events are not detectable using conventional tension probes. Each mutant peptide ligand displayed a different level of mechanical sampling and spatial scanning by the TCR that strongly correlated with its functional potency. Finally, we show evidence that T cells transmit pN forces through the programmed cell death receptor-1 (PD1), a major target in cancer immunotherapy. We anticipate that mechanical information storage will be broadly useful in studying the mechanobiology of the immune system.

Developing Nuclease-Resistant DNA-Based Tension Sensor for Cellular Force Imaging

Developing Nuclease-Resistant DNA-Based Tension Sensor for Cellular Force Imaging

The activation of IgM- or isotype-switched IgG- and IgE-BCR exhibits distinct mechanical force sensitivity and threshold.

B lymphocytes use B cell receptors (BCRs) to sense the physical features of the antigens. However, the sensitivity and threshold for the activation of BCRs resulting from the stimulation by mechanical forces are unknown. Here, we addressed this question using a double-stranded DNA-based tension gauge tether system serving as a predefined mechanical force gauge ranging from 12 to 56 pN. We observed that IgM-BCR activation is dependent on mechanical forces and exhibits a multi-threshold effect. In contrast, the activation of isotype-switched IgG- or IgE-BCR only requires a low threshold of less than 12 pN, providing an explanation for their rapid activation in response to antigen stimulation. Mechanistically, we found that the cytoplasmic tail of the IgG-BCR heavy chain is both required and sufficient to account for the low mechanical force threshold. These results defined the mechanical force sensitivity and threshold that are required to activate different isotyped BCRs.

Next Generation Mechanophores for Unraveling the Role of Integrin Tension in Focal Adhesion Assembly

Mechanical stimuli profoundly alter cell fate, yet the mechanisms underlying mechanotransduction remain obscure due to a lack of methods for molecular force imaging. In this presentation, I will describe the development of second and third generation mechanophores (molecules that fluoresce upon experiencing force) for imaging molecular forces exerted by individual integrin cell surface receptors. Specifically, we developed a new class of molecular tension probes that function as a switch to generate a 20-30-fold increase in fluorescence upon experiencing a threshold piconewton force. The probes employ immobilized DNA-hairpins with tunable force response thresholds, ligands, and fluorescence reporters. Quantitative imaging reveals that integrin tension is highly dynamic and increases with an increasing integrin density during adhesion formation. Mixtures of fluorophore-encoded probes show integrin mechanical preference for cyclized-RGD over linear-RGD peptides. Multiplexed probes with variable guanine-cytosine content within their hairpins reveal integrin preference for the more stable probes at the leading tip of growing adhesions near the cell edge. DNA-based tension probes are among the most sensitive optical force reporters to date, overcoming the force and spatial-resolution limitations of traction force microscopy. The application of these sensors to image forces associated with a range of mechano-regulatory processes that occur at the lipid membrane of the cell, such as endocytosis, Notch receptor activation, and integrin adhesion receptors will be described as well.

DNA-based digital tension probes reveal integrin forces during early cell adhesion.

Mechanical stimuli profoundly alter cell fate, yet the mechanisms underlying mechanotransduction remain obscure due to a lack of methods for molecular force imaging. Here, to address this need, we develop a new class of molecular tension probes that function as a switch to generate a 20–30-fold increase in fluorescence upon experiencing a threshold piconewton force. The probes employ immobilized DNA-hairpins with tunable force response thresholds, ligands, and fluorescence reporters. Quantitative imaging reveals that integrin tension is highly dynamic and increases with an increasing integrin density during adhesion formation. Mixtures of fluorophore-encoded probes show integrin mechanical preference for cyclized-RGD over linear-RGD peptides. Multiplexed probes with variable guanine-cytosine content within their hairpins reveal integrin preference for the more stable probes at the leading tip of growing adhesions near the cell edge. DNA-based tension probes are among the most sensitive optical force reporters to date, overcoming the force and spatial-resolution limitations of traction force microscopy.